CN106370850B - A kind of C.perfringens fluorescence immune chromatography detection kit and its application - Google Patents
A kind of C.perfringens fluorescence immune chromatography detection kit and its application Download PDFInfo
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Abstract
The present invention relates to a kind of C.perfringens fluorescence immune chromatography detection kit and its application, the kit includes fluorescence immune chromatography detection device and sample diluting liquid, the fluorescence immune chromatography detection device contains by bottom plate, sample pad, reagent pad, the test strips of nitrocellulose filter and absorption pad composition, the sample pad, reagent pad, nitrocellulose filter and absorption pad are staggeredly fixed on the bottom plate successively, the reagent pad is coated with the fluorescent microsphere of rabbit-anti C.perfringens IgG marks, detection line on nitrocellulose filter is coated with rabbit-anti C.perfringens IgG, nature controlling line is coated with goat anti-rabbit igg, the sample diluting liquid is the solution containing nano silver.The kit sensitivity is 10pg/L, and the range of linearity is 40~1000pg/L, and survey is time-consuming short, without special instruments and equipment and professional technician, can meet the needs that the units such as hospital, customs, health quarantine department are used for quickly detecting C.perfringens.
Description
Technical field
The invention belongs to which application field is immunized, it is related to the unit diagnostics such as medical institutions, epidemic prevention and quarantine department or detection aerogenesis
The technology of capsular clostridium, and in particular to a kind of C.perfringens fluorescence immune chromatography detection kit and its application.
Background technology
C.perfringens (C.perfringens), once claims clostridieum welchii or Bacillus perfringens, is that clinically gas is bad
Most common a kind of clostridium in subcutaneous ulcer pathogen, because that can decompose the sugar in muscle and connective tissue, produces a large amount of gases, causes tissue
Serious wind-puff, causes tissue large area downright bad, this bacterium can form pod membrane in vivo in addition, and therefore named aerogenesis presss from both sides film clostridium, is the mankind
The main pathogenic fungi of emphysematous gangrene.1892, American pathologists W.H.Welch etc. separated this bacterium from a corpse, thus also known as
Clostridieum welchii.There are 6 types of A~F according to toxin species and pathogenic difference, this bacterium is produced, wherein, some bacterial strains produce intestines
Toxin, can cause food poisoning.This bacterium is widely present in the excrement of soil, the enteron aisle of humans and animals and animals and humans,
Often infected because of deep wounds.In addition, C.perfringens can cause lamb dysentery and lamb, calf, piglet, rabbit, chick
Deng necrotic enteritis.At present, mainly include for the detection method of C.perfringens：Anaerobic bacteria culture, bacteria bio are special
Property identification, enzyme-linked immunosorbent assay (ELISA), PCR (PCR) etc., the consuming time length that these methods have,
Some conditions require height, and some is had a great influence by extraneous factor and weakens its practical value, received in terms of clinical detection
Very big limitation.Therefore, the diagnostic reagent of the quick detection C.perfringens of research, to carry out scene to gas gangrene infection
Detection and in time monitoring.
Immunochromatography is a kind of unique immunoassay method for coming across the eighties initial stage, due to widely using colloid
Gold is used as indicator, so the colloidal gold detection method that is otherwise known as.This method is widely used in sample primary dcreening operation at present, but shortcoming is inspection
It is relatively low to survey sensitivity, it is difficult to carry out quantitative detection.Fluorescent quantitation immunochromatography technique is immunofluorescence technique and traditional immunization layer
Analysis technology is combined a kind of quantitative new detection technique of development innovation, which is retaining colloidal gold immunochromatographimethod technical operation
Outside the advantages that simplicity, quick detection, also strengthening technology by fluorescent tracing realizes the accurate quantification of testing result.Fluorescent quantitation
Immunochromatography product is with the microsphere supported technology of functionalized nano, and with reference to fluorescent marker probe, instrument directly detects excitation fluorescence
Signal and CCD top layers densitometric scan technology used by non-traditional golden scalar quantity, therefore with higher signal-to-noise ratio, higher
Signal detection amount and detection sensitivity.But fluorescence signal intensity is linearly related to fluorescent microsphere quantity, fluorescent microsphere quantity
Preferable effect can just be had by needing to reach a certain amount of, and therefore, urgent need is developed a kind of without too many fluorescent microsphere, can also have very
The fluorescent quantitation immunochromatography technique of good effect, the detection for C.perfringens.
The content of the invention
In view of this, it is an object of the invention to：(1) a kind of C.perfringens fluorescence immune chromatography detection reagent is provided
Box；(2) a kind of method of C.perfringens fluorescence immune chromatography detection kit quantitative detection of clostridium perfringens.
To reach above-mentioned purpose, the present invention provides following technical solution：
1st, a kind of C.perfringens fluorescence immune chromatography detection kit, including fluorescence immune chromatography detection device and sample
Product dilution, the fluorescence immune chromatography detection device contain by bottom plate, sample pad, reagent pad, nitrocellulose filter and absorption
The test strips of composition are padded, the sample pad, reagent pad, nitrocellulose filter and absorption pad are staggeredly fixed on the bottom plate successively
On, the reagent pad is coated with the fluorescent microsphere of rabbit-anti C.perfringens IgG marks, the detection on the nitrocellulose filter
Line is coated with rabbit-anti C.perfringens IgG, and nature controlling line is coated with goat anti-rabbit igg, and the sample diluting liquid is to contain nanometer
The solution of silver.
Further, the concentration for the fluorescent microsphere that rabbit-anti C.perfringens IgG is marked is 1~2g/L on the reagent pad；
The concentration of rabbit-anti C.perfringens IgG is 1~2g/L in the detection line；The concentration of goat anti-rabbit igg on the nature controlling line
For 1~2g/L.
Further, the discharge rate for the fluorescent microsphere that rabbit-anti C.perfringens IgG is marked is 1~2 μ l/ on the reagent pad
cm；The discharge rate of rabbit-anti C.perfringens IgG is 1~2 μ l/cm in the detection line；Goat anti-rabbit igg on the nature controlling line
Discharge rate is 1~2 μ l/cm.
Further, the fluorescent microsphere is the polystyrene fluorescent microsphere of carboxylated.
Further, the fluorescence immune chromatography detection device further includes one and gets stuck, described to get stuck including panel and bottom plate, institute
Test strips are stated between the panel and bottom plate, well and detection hole, the well and inspection are provided with the panel
Gaging hole position respectively with reagent pad in the test strips and nitrocellulose filter position correspondence.
Further, the sample diluting liquid is made by following methods：
(1) preparation of Nano silver solution：Silver nitrate is added in deionized water, adds trisodium citrate after boiling by several times,
1min is boiled again after stirring the trisodium citrate dissolving added to last time, after being cooled to 25 DEG C, is settled to nanometer in solution
Silver concentration is 1~2mmol/L；
(2) preparation of sample diluting liquid：Obtained nanometer in step (1) is added into the buffer solution containing surfactant
Silver-colored solution, it is 0.1~0.2mmol/L to be settled to nanometer silver concentration in solution.
Further, in step (1), the mass ratio of the silver nitrate and the trisodium citrate added every time is 1:2~1:5；
The gradation is to be added once every 1min, totally 3 times.
Further, in step (2), the surfactant is Tween-20, the mass fraction of the Tween-20 for 1~
2%；The buffer solution is PBS buffer, and the pH value of the PBS buffer is 6.8~7.4.
2nd, a kind of method of C.perfringens fluorescence immune chromatography detection kit quantitative detection of clostridium perfringens, its
In, the kit is above-mentioned kit；The method is specially：Sample is mixed with sample diluting liquid in the kit
Afterwards, add and carry out immunochromatography reaction in the kit in fluorescence immune chromatography detection device, chromatograph after reaction through glimmering
Photodetector fluoroscopic examination.
Further, the volume ratio of the sample and sample diluting liquid is 1:10~1:15.
The beneficial effects of the present invention are：A kind of fluorescence immune chromatography the present invention provides C.perfringens detects examination
Agent box and its application, by the fluorescent microsphere concentration and discharge rate, rabbit-anti perfringens that determine rabbit-anti C.perfringens IgG marks
Clostridium IgG concentration and discharge rate, goat anti-rabbit igg concentration and discharge rate, assembling are prepared for the inspection of C.perfringens fluorescence immune chromatography
Device is surveyed, immunofluorescence quantitative measurement technology and nano silver surface plasma Fluorescence Increasing technology are employed, with the fluorescence immunoassay
Chromatography detection device coordinates the sample diluting liquid containing nano silver prepared in the present invention, the quantitative inspection for C.perfringens
Survey, has the following advantages：(1) high sensitivity, can reach 10pg/L (in terms of C.perfringens somatic antigen)；(2) linear model
Width is enclosed, can reach 40~1000pg/L；(3) high specificity, with this method with clostridium tetani, pseudomonas aeruginosa, large intestine
Escherichia, staphylococcus aureus, proteus vulgaris, 6 kinds of bacterium of enterococcus faecalis compare, wherein all bacterium use standard
Strain culturing, somatic antigen extraction, adjustment somatic antigen concentration are 100~200pg/L, and negative control is done with reagent background, its
Fluorescence intensity<100AU, the results show C.perfringens fluorescence intensity are 1500AU or so, are positive findings, other 6 kinds of bacterium
Fluorescence intensity be 100AU or so, be negative findings, show this test strips have well specificity；(4) this method surveys consumption
When it is short, it is only necessary to 10~15min, detection is easy to operate, without special instruments and equipment and professional technician, can meet hospital,
The needs that the units such as customs, health quarantine department are used for quickly detecting C.perfringens.
Brief description of the drawings
In order to make the purpose of the present invention, technical solution and beneficial effect clearer, the present invention provides drawings described below and carries out
Explanation：
Fig. 1 is the assembling schematic diagram of C.perfringens fluorescence immune chromatography test paper bar；
Fig. 2 detects card schematic diagram for C.perfringens fluorescence immune chromatography；
Fig. 3 is the detection principle diagram of perfringens reagent fluorescent immune method；
Fig. 4 is the transmission electron microscope picture of Nano silver solution；
Fig. 5 is the Fluorescence Increasing performance analysis chart of nano-Ag particles；
Fig. 6 is the sensitivity test figure of C.perfringens fluorescence immune chromatography detection kit；
Fig. 7 is the range of linearity test chart of C.perfringens fluorescence immune chromatography detection kit；
Fig. 8 is the specific test figure of C.perfringens fluorescence immune chromatography detection kit；
Embodiment
Below in conjunction with attached drawing, the preferred embodiment of the present invention is described in detail.
Material and reagent：Polystyrene fluorescent microsphere (article No.：L050L), it is purchased from Guangzhou ten thousand and inspires confidence in biotech company；1-
(3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides, n-hydroxysuccinimide, bovine serum albumin(BSA), Tween-20,
Trehalose, silver nitrate, are purchased from Sigma-Aldrich；Polyester fiber film, is purchased from Nanjing micrometering biotechnology company；
Trisodium citrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, are purchased from West Asia Reagent Company；Rabbit-anti C.perfringens IgG (article No.s：
Ab35023), goat anti-rabbit igg (article No.：Ab6702), it is purchased from Abcam companies of the U.S.；Gas capsular clostridium somatic antigen standard items
(bacterium source：American Type Culture collection warehousing, American type culture collection, ATCC；Numbering：
ATCC13124), clostridium tetani, pseudomonas aeruginosa, escherichia coli, staphylococcus aureus, proteus vulgaris,
Enterococcus faecalis (bacterium derives from U.S. ATCC)；FIC-S2011-E2 fluorescence immune chromatography readout instruments, Shanghai fly to survey biotechnology
Company produces.
First, the preparation of the fluorescence immune chromatography detection card of C.perfringens
(1) preparation of the fluorescent microsphere and reagent pad of rabbit-anti C.perfringens IgG marks
Polystyrene fluorescent microsphere of the 1.0ml concentration for 1g/L carboxylated is taken, adds 20mg 1- (3- dimethylaminos third
Base) -3- ethyl-carbodiimide hydrochlorides (EDC) and 12mg n-hydroxysuccinimides (NHS), 30min is incubated at 37 DEG C, so
1min, supernatant discarding are being centrifuged with 10000r/min 4 DEG C afterwards, precipitation is resuspended with 1.0ml PBS, is repeated resuspension-centrifugation-and is removed supernatant
It must be precipitated after 3 times, the C.perfringens antibody for adding that 200 μ l concentration are 1.0g/L is precipitated to gained, it is anti-at 25 DEG C after mixing
Answer 3h；Then 10 μ l, 10% bovine serum albumin(BSA)s (BSA) are added, 1.5h is incubated at 37 DEG C, close fluorescent microsphere；Finally at 4 DEG C
Under 5min, supernatant discarding centrifuged with 10000r/min, precipitation is resuspended with 1.0ml PBS, is repeated after resuspension-centrifugation-removes supernatant 3 times
It must precipitate, precipitate and be resuspended with the 1.0ml PBS solutions for containing 1%BSA, 2%Tween-20 and 25% trehalose, as prepared
Rabbit-anti C.perfringens IgG mark fluorescent microsphere solution, 4 DEG C save backup.
Polyester fiber film mass fraction is soaked into 10min for 0.1% Tween-20,60 DEG C of exhausting drying, are by concentration
The fluorescent microsphere of the rabbit-anti C.perfringens IgG marks of 1.5g/L is sprayed on polyester fiber film by 1.5 μ l/cm, 37 DEG C of dryings
After 1h, reagent pad is made, is sealed under 25 DEG C of dry environments.
(2) on nitrocellulose filter detection line and nature controlling line spray
The rabbit-anti C.perfringens IgG that concentration is 1.5g/L is sprayed on cellulose nitrate by 1 μ l/cm using automatic spray film instrument
Detection line (T lines) is formed on plain film (NC films), the goat anti-rabbit igg that concentration is 1.5g/L is sprayed on nitrocellulose by 1 μ l/cm
Nature controlling line (C lines) is formed on film, after 37 DEG C of dry 1h, is sealed under 25 DEG C of dry environments.
(3) assembling of detection card
NC films are cut into 17mm wide with cutting machine, are pasted at the center of plastic bottom board.Then, reagent pad is cut into
10mm wide, overlap joint paste the T line ends in NC films, and the width of lap is 2mm, then blotting paper is cut into 17mm wide, overlap joint
The C line ends in NC films are pasted, the width of lap is 2mm.Finally, sample pad is cut into 12mm wide, overlap joint, which is pasted, to be tried
The sample end of agent pad, the width of lap is 2mm, and assembled formation (such as Fig. 1), wherein the distance between T lines and C lines are 5mm,
T lines and C line-spacing NC film edges 6mm.Taken out after test strip is placed in 37 DEG C of temperature-controlled box drying 1h, be cut into the test paper of 4mm wide
Bar, test strips is fitted into getting stuck (such as Fig. 2), is sealed under 25 DEG C of dry environments.Wherein, this gets stuck including panel and bottom
Plate, is provided with well, detection hole and convex shelves on panel, the position of well and detection hole respectively with immuno-chromatographic test paper strip
The position correspondence of reagent pad and NC films, convex shelves are arranged on operating side of getting stuck, and facilitate insertion into detector and taking-up, and bottom plate is provided with card
Groove tries, for placing test paper.
2nd, the preparation of the dilution containing nano silver
(1) preparation of Nano silver solution
Weigh 0.2g silver nitrates to add in 500ml deionized waters, by 1 time/min, the mode point of 1.0g/ times 3 times after boiling
Trisodium citrate is added, 1min is boiled again after stirring the trisodium citrate dissolving added to last time, after being cooled to 25 DEG C, adds
Deionized water is to 1000ml, and nanometer silver concentration is 1mmol/L in solution, and the Nano silver solution applied in the present invention, 4 DEG C of guarantors are made
Deposit spare.
1) transmission electron microscope analysis of nano-Ag particles
100 μ l Nano silver solutions are taken, are added dropwise on transmission electron microscope (TEM) dedicated test copper mesh, dry, Tecnai
G2F20S-TWIN (200KV) types transmission electron microscope detects, shown in testing result Fig. 4, as shown in Figure 4, obtained nano silver
Granule-morphology almost spherical, particle diameter distribution is uniform, between 50~70nm, agglomeration, favorable dispersibility does not occur.
2) the Fluorescence Increasing performance evaluation of nano-Ag particles
1. sample is grouped：a：Nano silver solution (0.5ml sample+0.5ml water)；b：Polystyrene fluorescent microsphere (0.5ml samples
Product+0.5ml water)；c：Nano silver solution (0.5ml) and polystyrene fluorescent microsphere (0.5ml) mixed liquor.
It is detected in Hitachi 2. (Hitachi) F-7000 type Fluorescence Spectrometer.Excitation wavelength：365nm, fluoroscopic examination ripple
It is long：610nm.Each sample is surveyed 5 times.
Testing result is as shown in figure 5, as shown in Figure 5, under 365nm excitation wavelengths, the fluorescent absorption peak of Nano silver solution
Intensity is minimum, no obvious fluorescence interference.After adding Nano silver solution, the fluorescence intensity ratio of polystyrene fluorescent microsphere mixed liquor
The fluorescence intensity of simple polystyrene fluorescent microsphere substantially increases, about 2.5~3.0 times, the nano silver for prompting the present invention to prepare
The Fluorescence Increasing significant effect of solution.
(2) preparation of sample diluting liquid
Weigh 71.6g disodium hydrogen phosphates and 31.2g sodium dihydrogen phosphates are added in 500ml deionized waters, be made into pH value as 6.8
PBS buffer, Tween-20 and the above-mentioned nano silvers of 100ml that 20ml mass fractions are 2% are then added into gained buffer solution
Solution, adds deionized water to 1000ml, and nanometer silver concentration is 0.1mmol/L in solution, and the sample diluting liquid in the present invention is made,
4 DEG C save backup.
3rd, the fluorescence immune chromatography detection kit of C.perfringens is qualitative and quantitative detection of clostridium perfringens
The fluorescence immune chromatography detection kit of C.perfringens is qualitative and the principle of quantitative detection of clostridium perfringens
As shown in figure 3, sample to be detected is detected with after sample diluting liquid mixes in kit, taking mixed solution to instill in kit
Well on card, mixed solution is after sample pad filtering in test strips, C.perfringens somatic antigen and examination in sample
The fluorescent microsphere of rabbit-anti C.perfringens IgG marks in agent pad combines, and is first produced by chromatography effect with the rabbit-anti on NC films
Gas capsular clostridium IgG forms detection line (T lines), the rabbit-anti perfringens shuttle being not associated with after combining to form double antibodies sandwich compound
The fluorescent microsphere of bacterium IgG marks continues chromatography and is combined with goat anti-rabbit igg, forms nature controlling line (C lines).Meanwhile sample diluting liquid
In nano-Ag particles also synchronously carried out immunochromatography, and arrive separately at T lines and C line positions.
1) the fluorescence immune chromatography detection kit qualitative detection C.perfringens of C.perfringens
1. if shows green fluorescence trace, expression testing result are sun at the same time under uv excitation light irradiation for T lines and C lines
Property, illustrate to be detected in sample and contain C.perfringens somatic antigen.
If 2. the T line redgreen fluorescence traces under uv excitation light irradiation, C line shows green fluorescence traces, represent detection
As a result it is feminine gender, illustrates to be free of C.perfringens somatic antigen in detected sample, or its concentration is less than 10pg/L.
If 3. the C line redgreen fluorescence traces under uv excitation light irradiation, this detection is invalid, need to analyze failure cause simultaneously
Again detect.If fluorescence immune chromatography detection card is used in conjunction, it can be achieved that quantitative detection with fluorescent quantitative detector.
2) the fluorescence immune chromatography detection kit quantitative detection of clostridium perfringens of C.perfringens
1. fluorogenic quantitative detection：Excitation wavelength：365nm；Fluoroscopic examination wavelength：610nm.
2. the measure of calibration curve equation formula：The C.perfringens sample of gradient concentration：Take C.perfringens thalline
Antigen standard, compound concentration are respectively 1,2,4,8,16,32,64,128,256,612,1024pg/L sample.Sample-adding：Point
20 μ l samples are not taken, are added in 250 μ l sample diluting liquids, are mixed, and take 100 μ l mixed liquors to be added dropwise in C.perfringens after 1min
Fluorescence immune chromatography detection card.Immunochromatography：Immunochromatographydetection detection card after sample-adding places 5min at 25 DEG C, carries out immune layer
Analysis.Fluoroscopic examination：Immunofluorescence analysis is carried out using fluorescence detector.Each sample surveys second-order fluorescence, is averaged.Standard is bent
The formulation of line：Sample concentration and the fluorescent value of detection are subjected to linear regression analysis, obtain calibration curve equation formula.
3. C.perfringens quantitatively detects：Sample-adding：20 μ l samples are taken respectively, are added in 250 μ l sample diluting liquids, are mixed
It is even, take 100 μ l mixed liquors to be added dropwise on C.perfringens fluorescence immune chromatography detection card after 1min.Immunochromatography：After sample-adding
Immunochromatographydetection detection card place 5min at 25 DEG C, carry out immunochromatography.Fluoroscopic examination：Using fluorescence immune chromatography readout instrument
Carry out fluorescence analysis.Each sample surveys second-order fluorescence, is averaged.Quantifying reporter result：The fluorescent value of sample detection is substituted into
In calibration curve equation formula, C.perfringens concentration is obtained, repetition is surveyed secondary, is averaged report result.
4th, the performance evaluation of the fluorescence immune chromatography detection kit of C.perfringens
1) sensitivity test
Sample is detected according to 100,80,60,40,20,10,8,6,4,2,1pg/L gradient dilutions, takes 20 μ l samples respectively,
Add in 250 μ l sample diluting liquids, mix, take 100 μ l mixed liquors to be added dropwise in C.perfringens fluorescence immune chromatography after 1min
On detection card, carry out immunochromatography and fluorescence analysis is carried out using fluorescence immune chromatography readout instrument, each sample repeats detection 5
It is secondary.Test result is as shown in fig. 6, it will be appreciated from fig. 6 that the C.perfringens of 10pg/L concentrations above has obvious fluorescence signal
(100~500AU), then fluorescence signal declines rapidly (20~50AU) below 10pg/L concentration.According to reference method test limit
(LOD, Limit of detection) is the respective amounts of 3 times of instrumental background signal caused by matrix blank values, this detection
The lowest detection of method is limited to 10pg/L or so.
2) range of linearity is tested
Sample is detected according to 2000,1000,500,250,125,70,40,20,10pg/L gradient dilutions, takes 20 μ l respectively
Sample, adds in 250 μ l sample diluting liquids, mixes, and takes 100 μ l mixed liquors to be added dropwise in C.perfringens fluorescence immunoassay after 1min
On chromatography detection card, carry out immunochromatography and fluorescence analysis is carried out using fluorescence immune chromatography readout instrument, each sample repeats to examine
Survey 5 times.Test result is as shown in fig. 7, as shown in Figure 7, the C.perfringens of 10~2000pg/L gradient concentrations has accordingly
Fluorescence signal (100~5000AU), wherein then fluorescence signal declines rapidly below 40pg/L concentration, 1000pg/L concentrations above
Then fluorescence signal climbing speed substantially slows down (Fig. 7 a).According to the coefficient R of equation of linear regression>0.95(R2>0.90) count,
The range of linearity of this detection method is 40~1000pg/L (Fig. 7 b).
3) specific test
1. sample is grouped：Negative control, C.perfringens, 6 kinds of wound infection encountered pathogenic bacterias (clostridium tetani, copper
Green pseudomonad, escherichia coli, staphylococcus aureus, proteus vulgaris, enterococcus faecalis).All bacterium are using mark
Quasi- strain culturing, somatic antigen extraction, adjustment somatic antigen concentration are 100~200pg/L.
2. taking 20 μ l samples respectively, add in 250 μ l sample diluting liquids, mix, take 100 μ l mixed liquors to be added dropwise after 1min
On C.perfringens fluorescence immune chromatography detection card, carry out immunochromatography and fluorescence is carried out using fluorescence immune chromatography readout instrument
Analysis, each sample repeat detection 5 times.
Testing result is as shown in figure 8, as shown in Figure 8, negative control fluorescence intensity<100AU, is reagent background；Aerogenesis pod
Film clostridium fluorescence intensity is 1500AU or so, is positive findings；The fluorescence intensity of other 6 kinds of bacterium is 100AU or so, for feminine gender
As a result.The results show that this fluoroscopic examination card and wound infection encountered pathogenic bacteria have good specificity without obvious cross reaction.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
Cross above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Claims (8)
1. a kind of C.perfringens fluorescence immune chromatography detection kit, including fluorescence immune chromatography detection device and sample it is dilute
Liquid is released, the fluorescence immune chromatography detection device contains by bottom plate, sample pad, reagent pad, nitrocellulose filter and absorption pad group
Into test strips, the sample pad, reagent pad, nitrocellulose filter and absorption pad successively staggeredly is fixed on the bottom plate, institute
State the fluorescent microsphere that reagent pad is coated with rabbit-anti C.perfringens IgG marks, the detection line bag on the nitrocellulose filter
There is rabbit-anti C.perfringens IgG, nature controlling line is coated with goat anti-rabbit igg, and the sample diluting liquid is to contain nano silver
Solution；The concentration for the fluorescent microsphere that rabbit-anti C.perfringens IgG is marked is 1 ~ 2 g/L on the reagent pad；The detection line
The concentration of upper rabbit-anti C.perfringens IgG is 1 ~ 2 g/L；The concentration of goat anti-rabbit igg is 1 ~ 2 g/L on the nature controlling line；
The discharge rate for the fluorescent microsphere that rabbit-anti C.perfringens IgG is marked is 1 ~ 2 μ l/cm on the reagent pad；Rabbit in the detection line
The discharge rate of anti-C.perfringens IgG is 1 ~ 2 μ l/cm；The discharge rate of goat anti-rabbit igg is 1 ~ 2 μ l/cm on the nature controlling line.
2. a kind of C.perfringens fluorescence immune chromatography detection kit as claimed in claim 1, it is characterised in that described
Fluorescent microsphere is the polystyrene fluorescent microsphere of carboxylated.
3. a kind of C.perfringens fluorescence immune chromatography detection kit as claimed in claim 1, it is characterised in that described
Fluorescence immune chromatography detection device further includes one and gets stuck, described to get stuck including panel and bottom plate, and the test strips are located at the face
Between plate and bottom plate, be provided with well and detection hole on the panel, the well and detection hole site respectively with it is described
Reagent pad and nitrocellulose filter position correspondence in test strips.
4. a kind of C.perfringens fluorescence immune chromatography detection kit as claimed in claim 1, it is characterised in that described
Sample diluting liquid is made by following methods：
（1）The preparation of Nano silver solution：Silver nitrate is added in deionized water, adds trisodium citrate, stirring after boiling by several times
1 min is boiled again after the trisodium citrate dissolving added to last time, and after being cooled to 25 DEG C, it is dense to be settled to nano silver in solution
Spend for 1 ~ 2 mmol/L；
（2）The preparation of sample diluting liquid：Step is added into the buffer solution containing surfactant（1）In obtained nano silver it is molten
Liquid, it is 0.1 ~ 0.2 mmol/L to be settled to nanometer silver concentration in solution.
A kind of 5. C.perfringens fluorescence immune chromatography detection kit as claimed in claim 4, it is characterised in that step
（1）In, the mass ratio of the silver nitrate and the trisodium citrate added every time is 1:2~1:5；The gradation is to add every 1 min
Enter once, totally 3 times.
A kind of 6. C.perfringens fluorescence immune chromatography detection kit as claimed in claim 4, it is characterised in that step
（2）In, the surfactant is Tween-20, and the mass fraction of the Tween-20 is 1 ~ 2%；The buffer solution buffers for PBS
Liquid, the pH value of the PBS buffer is 6.8 ~ 7.4.
7. a kind of method of C.perfringens fluorescence immune chromatography detection kit quantitative detection of clostridium perfringens, its feature
It is, the kit is claim 1 ~ 6 any one of them kit；The method is specially：By sample and the examination
In agent box after sample diluting liquid mixing, add in the kit in fluorescence immune chromatography detection device that to carry out immunochromatography anti-
Should, chromatograph after reaction through fluorescence detector fluoroscopic examination.
8. a kind of C.perfringens fluorescence immune chromatography detection kit as claimed in claim 7 quantitatively detects perfringens
The method of clostridium, it is characterised in that the volume ratio of the sample and sample diluting liquid is 1:10~1:15.
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| CN111024946A (en) * | 2019-11-19 | 2020-04-17 | 江苏美克医学技术有限公司 | Trichomonas vaginalis fluorescence immunochromatography assay kit and preparation method thereof |
| CN113238044A (en) * | 2021-01-27 | 2021-08-10 | 中国人民解放军军事科学院军事医学研究院 | Rapid and quantitative detection card for clostridium perfringens epsilon toxin |
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| CN104237537A (en) * | 2014-10-21 | 2014-12-24 | 南京基蛋生物科技有限公司 | Immune chromatography fluorescence reagent strip for detecting cardiac troponin and preparation method thereof |
| CN104897906A (en) * | 2015-06-25 | 2015-09-09 | 浙江大学 | Dual quantitative protein toxin quantum dot fluorescence immunochromatographic strip and preparation method thereof |
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| CN104237537A (en) * | 2014-10-21 | 2014-12-24 | 南京基蛋生物科技有限公司 | Immune chromatography fluorescence reagent strip for detecting cardiac troponin and preparation method thereof |
| CN104897906A (en) * | 2015-06-25 | 2015-09-09 | 浙江大学 | Dual quantitative protein toxin quantum dot fluorescence immunochromatographic strip and preparation method thereof |
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